User:Karmella Haynes/Notebook/Polycomb project/2010/06/02

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06/02/10

  • ✓ KAH130-2: thawed cells barely growing; some colonies; refresh 20% FBS medium
  • ✓ Senescence assay: plan optimization
  • ✓ Growth assay: split cell lines 1:10 in 10 cm plates; 1 +ab's, 1 plain med., 1 plain med. + 1μg/mL dox (refresh every 2 days) (starting over, throw out old overgrown plates)



Senescence assay
> Flow cytometry based method using C12-FDG; based on Noppe et al. (Cytometry, 2009) and Kurz et al. (J or Cell Sci, 2000)
--> pH modulation; appears to be essential only for X-gal method (not C12-FDG)
--> Step 2: Rotenone treatment (senescence inducer); working concentration = 0.2 μg/mL (~0.6 μM), >3 days
--> Step 3: C12-FDG treatment; working concentration = 30 μg/mL (~33 μM), 1 hour
--> Step 4: visualize w/ FITC microscope setting or harvest for flow cytometry (500-510 nm wavelength)

> Stock solutions
--> 50x C12-FDG: 5 mg C12-FDG in 3.3 mL 70% DMSO/PBS* (1.5 mg/mL); Aliquot and store @ -20°C
--> 10,000x rotenone: 10 mg rotenone in 5 mL 100% DMSO (2.0 mg/mL); make 1000x rotenone, aliquot, store @ -20°C

*Accidentally added 1 mL PBS before realizing C12-FDG is not soluble in H2O. Solubility appears okay in 70% DMSO.